A pyridine derivative, intermediates, processes for their preparation and uses

Abstract

The application discloses a pyridine derivative, an intermediate, a preparation method and application. Specifically disclosed is a compound as shown in formula I or a pharmaceutically acceptable salt thereof. The pyridine derivative of the application can inhibit the growth of sensitive Mycobacterium tuberculosis, drug-resistant Mycobacterium tuberculosis, and in particular, bedaquiline drug-resistant strains, and has the advantages of longer elimination half-life in pharmacokinetics and higher safety.

Description

TECHNICAL FIELD

[0001] The present application relates to a pyridine derivative, an intermediate, a preparation method and an application. BACKGROUND

[0002] Pulmonary tuberculosis (PTB) refers to tuberculosis lesions occurring in lung tissue, trachea, bronchus and pleura.

[0003] Bedaquiline is a diarylquinoline derivative, which was approved for marketing in the United States in December 2012, in the European Union in March 2014, and in China in November 2016. Bedaquiline (BDQ) was listed as the first choice for rifampicin-resistant tuberculosis (RR-TB) and multidrug-resistant tuberculosis (MDR-TB) by the World Health Organization (WHO) in 2018.

[0004] The patent with application number 201410335196.X records a pyridine derivative and its application as an anti-mycobacterium, which is used for treating pulmonary tuberculosis and multidrug-resistant pulmonary tuberculosis.

[0005] Complications of infectious tuberculosis cause multidrug-resistant tuberculosis. Globally, 4% of cases are associated with multidrug-resistant tuberculosis.

[0006] The proportion of multidrug-resistant tuberculosis population remains at about 3-4% in the initial diagnosis, and about 18-21% in patients with treatment experience. Drug-resistant tuberculosis is still a public health threat. The current long and complex treatment regimen is far from sufficient to curb and defeat multidrug-resistant tuberculosis infection, thus prompting people to find better drugs. SUMMARY

[0007] The technical problem to be solved by the present application is to overcome the drug resistance problem of tuberculosis in the prior art, and to provide a pyridine derivative and an application. The pyridine derivative of the present application can inhibit the growth of sensitive Mycobacterium tuberculosis, drug-resistant Mycobacterium tuberculosis, and especially bedaquiline-resistant strains, and has the advantages of longer half-life elimination in pharmacokinetics and high safety.

[0008] The present application provides a compound as shown in formula I or a pharmaceutically acceptable salt thereof;

[0009]

[0010] I.

[0011] The present application also provides a compound as shown in formula I-X or a pharmaceutically acceptable salt thereof;

[0012]

[0013] I-X.

[0014] The present application also provides a compound as shown in formula I-Y or a pharmaceutically acceptable salt thereof.

[0015]

[0016] I-Y;

[0017] The retention time of the compound as shown in formula I-Y under the following SFC conditions is 1.445 min.

[0018] Column: Chiralpak AD-3.50A, 4.6mm, inner diameter 3um;

[0019] Mobile phase: A: CO2, B: EtOH (0.05% diethanolamine);

[0020] Gradient: MPB (mobile phase B) from 5% to 50% in 1.2 min, hold for 1 min, then 0.8 min from 50% to 5%;

[0021] Flow rate: 3.4 mL / min;

[0022] Column temperature: 35℃;

[0023] ABPR: 1800 psi.

[0024] The present application also provides a compound as shown in formula II,

[0025]

[0026] II;

[0027] R1 is an amino protecting group.

[0028] In one embodiment, R1 is preferably -Bn, -Trt, -Dmb or -PMB, more preferably -Bn.

[0029] The present application also provides a compound as shown in formula II-X,

[0030]

[0031] II-X;

[0032] R1 is as defined above.

[0033] The present application also provides a preparation method of a compound as shown in formula I, comprising the following steps: performing a deprotection reaction on a compound as shown in formula II-X in a solvent in the presence of a deprotection reagent, and then performing chiral resolution to obtain a compound as shown in formula I.

[0034] ;

[0035] wherein R1is as defined above.

[0036] In one embodiment, the method and conditions of the deprotection reaction can be conventional for such reactions in the art.

[0037] In one embodiment, the solvent can be conventional for such reactions in the art, for example, dichloromethane.

[0038] In one embodiment, the amount of the solvent can be conventional for such reactions in the art, for example, the volume to mass ratio of the solvent to the compound of formula II-X can be 3 mL / g ~ 6 mL / g, preferably 5 mL / g, 4 mL / g or 4.7 mL / g.

[0039] In one embodiment, the deprotection reagent can be conventional for such reactions in the art, for example, chloroformic acid-1-chloroethyl ester and methanol.

[0040] In one embodiment, the amount of the deprotection reagent can be conventional for such reactions in the art, for example, the molar ratio of the deprotection reagent to the compound of formula II-X can be 1.1:1 ~ 1.8:1, preferably 1.4:1.

[0041] In one embodiment, the temperature of the deprotection reaction can be conventional for such reactions in the art, for example, room temperature.

[0042] In one embodiment, the progress of the deprotection reaction can be monitored by conventional detection methods for such reactions in the art, for example, TLC, HPLC, GC or NMR, preferably HPLC.

[0043] In one embodiment, the deprotection reaction can further comprise the following post-treatment steps: after the completion of the deprotection reaction is monitored, the reaction is quenched, extracted and separated after the PH is adjusted, the obtained organic phase is concentrated and dried, and then separated by column chromatography to obtain the compound of formula I-X.

[0044] The present application also provides a preparation method of a compound of formula I, which comprises the following steps: in a solvent, the compound of formula II is subjected to a deprotection reaction in the presence of a deprotection reagent to obtain the compound of formula I;

[0045] ;

[0046] wherein R1is as defined above.

[0047] In an embodiment, the method and conditions of the deprotection reaction can be conventional for such reactions in the art.

[0048] In an embodiment, the solvent can be conventional for such reactions in the art, for example, dichloromethane.

[0049] In an embodiment, the amount of solvent used can be conventional for such reactions in the art, for example, the volume to mass ratio of the solvent to the compound of formula II can be 3 mL / g to 6 mL / g, preferably 5 mL / g, 4 mL / g or 4.7 mL / g.

[0050] In an embodiment, the deprotection reagent can be conventional for such reactions in the art, for example, chloroformic acid-1-chloroethyl ester and methanol.

[0051] In an embodiment, the amount of deprotection reagent used can be conventional for such reactions in the art, for example, the molar ratio of the deprotection reagent to the compound of formula II can be 1.1:1 to 1.8:1, preferably 1.4:1.

[0052] In an embodiment, the temperature of the deprotection reaction can be conventional for such reactions in the art, for example, room temperature.

[0053] In an embodiment, the progress of the deprotection reaction can be monitored using conventional detection methods for such reactions in the art, for example, TLC, HPLC, GC or NMR, preferably HPLC.

[0054] In an embodiment, the deprotection reaction can further comprise the following post-treatment steps: after the completion of the deprotection reaction is monitored, the reaction is quenched, extracted and separated after the PH is adjusted, the obtained organic phase is concentrated and dried, and then separated by column chromatography to obtain the compound of formula I.

[0055] The present application also provides a method for preparing a compound of formula II-X, which comprises the following steps: in a solvent, a compound of formula III is reacted with a compound of formula IV as shown below to obtain the compound of formula II-X.

[0056] ;

[0057] wherein R1 is as defined above.

[0058] In an embodiment, the method and conditions of the above reaction can be conventional for such reactions in the art.

[0059] In an embodiment, the solvent can be conventional for such reactions in the art, for example, tetrahydrofuran.

[0060] In an embodiment, the solvent can be used in an amount conventional in the art for such reactions, for example, the volume to mass ratio of the solvent to the compound of formula III can be 4 mL / g to 6 mL / g, preferably 5 mL / g.

[0061] In an embodiment, the base can be a base conventional in the art for such reactions, for example, n-butyllithium.

[0062] In an embodiment, the base can be used in an amount conventional in the art for such reactions, for example, the molar ratio of the base to the compound of formula III can be 1.2:1 to 2:1, preferably 1.65:1.

[0063] In an embodiment, the temperature of the deprotection reaction can be a temperature conventional in the art for such reactions, for example, -60°C to -80°C, preferably -72°C.

[0064] In an embodiment, the progress of the above reaction can be monitored using a method conventional in the art for such reactions.

[0065] In an embodiment, the method for preparing the compound of formula II-X, the post-treatment step can be a post-treatment step conventional in the art for such reactions, for example, comprising the following steps: after the reaction is monitored to be completed, the obtained reaction solution is added into a saturated NH4Cl solution, washed, extracted and separated, the obtained organic phase is washed with brine and separated, the obtained organic phase is concentrated, dried and separated by column chromatography to obtain the compound of formula II-X.

[0066] The present application also provides use of substance A or substance B in the preparation of a drug for inhibiting Mycobacterium tuberculosis; the substance A is a compound of formula I or a pharmaceutically acceptable salt thereof; the substance B is a compound of formula I-X or a pharmaceutically acceptable salt thereof.

[0067] In an embodiment, the Mycobacterium tuberculosis is preferably sensitive Mycobacterium tuberculosis or drug-resistant Mycobacterium tuberculosis.

[0068] In an embodiment, the sensitive Mycobacterium tuberculosis is preferably strain / ATCC 27830.

[0069] In an embodiment, the drug-resistant Mycobacterium tuberculosis is preferably bedaquiline-resistant Mycobacterium tuberculosis; the bedaquiline-resistant Mycobacterium tuberculosis is preferably selected from one or more of BDQ-resistant strain 1, BDQ-resistant strain 2 and BDQ-resistant strain 3.

[0070] In some embodiments, the drug can comprise a pharmaceutical adjuvant.

[0071] In some embodiments, the substance A or the substance B can be one or the only effective component of the drug.

[0072] In some embodiments, the substance A is preferably a compound as shown in formula I.

[0073] In some embodiments, the substance B is preferably a compound as shown in formula I-X.

[0074] In some embodiments, the drug can be administered in a manner conventional in the art, for example orally.

[0075] In some embodiments, the drug can be administered at a frequency conventional in the art, preferably once a week.

[0076] The present application also provides use of a substance A or a substance B in the preparation of a drug for treating Mycobacterium tuberculosis infection; the substance A is a compound as shown in formula I or a pharmaceutically acceptable salt thereof; the substance B is a compound as shown in formula I-X or a pharmaceutically acceptable salt thereof.

[0077] In an embodiment, the Mycobacterium tuberculosis is preferably sensitive Mycobacterium tuberculosis or drug-resistant Mycobacterium tuberculosis.

[0078] In an embodiment, the drug-resistant Mycobacterium tuberculosis is preferably strain / ATCC 27830.

[0079] In an embodiment, the drug-resistant Mycobacterium tuberculosis is preferably bedaquiline-resistant Mycobacterium tuberculosis; the bedaquiline-resistant Mycobacterium tuberculosis is preferably selected from one or more of BDQ-resistant strain 1, BDQ-resistant strain 2 and BDQ-resistant strain 3.

[0080] In some embodiments, the drug can comprise pharmaceutical adjuvants.

[0081] In some embodiments, the disease of Mycobacterium tuberculosis infection is preferably pulmonary tuberculosis.

[0082] In some embodiments, the substance A or the substance B can be one or the only effective component of the drug.

[0083] In some embodiments, the substance A is preferably a compound as shown in formula I.

[0084] In some embodiments, the substance B is preferably a compound as shown in formula I-X.

[0085] In some embodiments, the drug can be administered in a manner conventional in the art, for example orally.

[0086] In some embodiments, the pharmaceutical is administered at a frequency that is conventional in the art, preferably once per week.

[0087] The present application also provides a pharmaceutical composition comprising Substance A or Substance B, and a pharmaceutically acceptable excipient; the Substance A is a compound of Formula I or a pharmaceutically acceptable salt thereof; the Substance B is a compound of Formula I-X or a pharmaceutically acceptable salt thereof.

[0088] In the present application, the compound of Formula I-X is a mixture of chiral isomers comprising at least the compound of Formula I, unless otherwise specified.

[0089] In the present application, the "room temperature" is 20-30°C, preferably 25°C, unless otherwise specified.

[0090] In the present application, the terms have the following meanings, unless otherwise specified:

[0091] The term "pharmaceutically acceptable" means that the salt, solvent, excipient, etc. is generally non-toxic, safe, and suitable for use with patients. The "patient" is preferably a mammal, more preferably a human.

[0092] The term "pharmaceutically acceptable salt" means a salt prepared from a compound of the present application with a relatively nontoxic, pharmaceutically acceptable acid or base. When a compound of the present application contains relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the pharmaceutically acceptable base in pure form or in solution in a suitable inert solvent. When a compound of the present application contains relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the pharmaceutically acceptable acid in pure form or in solution in a suitable inert solvent. The pharmaceutically acceptable acids include both inorganic acids and organic acids. When a compound of the present application contains both relatively acidic and relatively basic functionalities, acid and base addition salts can be formed. See, e.g., Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66: 1-19 (1977), or Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl and Camille G. Wermuth, ed., Wiley-VCH, 2002).

[0093] The term "pharmaceutically acceptable excipient" refers to all substances, other than the active pharmaceutical ingredient, contained in a pharmaceutical preparation, which are generally divided into two categories: excipients and additives. For details, please refer to the "People's Republic of China Pharmacopoeia (2020 Edition)", Handbook of Pharmaceutical Excipients (Paul J Sheskey, Bruno C Hancock, Gary P Moss, David J Goldfarb, 2020, 9th Edition).

[0094] The term "amino protecting group" refers to a protecting group suitable for preventing side reactions on the amino nitrogen. Representative amino protecting groups include, but are not limited to, arylmethyl groups such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), trityl (Trt) and 2.4-dimethoxybenzyl (Dmb).

[0095] Without deviating from the common sense of the art, the above-mentioned preferred conditions can be combined arbitrarily, i.e. to obtain each preferred example of the present application.

[0096] The compounds of the present application can be prepared by various synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by the combination of the specific embodiments with other chemical synthetic methods well known to those skilled in the art, and equivalent replacement methods well known to those skilled in the art, preferred embodiments including but not limited to the examples of the present application.

[0097] The following abbreviations are used in the present application: aq represents water; eq represents equivalent, equivalent amount; DCM represents dichloromethane; EtOAc represents ethyl acetate; EtOH represents ethanol; MeOH represents methanol; Bn represents benzyl; PMB represents p-methoxybenzyl; Trt represents trityl; and Dmb represents 2.4-dimethoxybenzyl.

[0098] The reagents and raw materials used in the present application are commercially available.

[0099] The positive progress effect of the present application is that: (1) the pyridine derivative of the present application can inhibit the growth of sensitive Mycobacterium tuberculosis, drug-resistant Mycobacterium tuberculosis and bedaquiline-resistant strains;

[0100] (2) the pyridine derivative of the present application has a longer elimination half-life in pharmacokinetics;

[0101] (3) the pyridine derivative of the present application has higher safety. DETAILED DESCRIPTION

[0102] The application will be further described in the following by way of examples without limiting the application to the examples described. The experimental methods in the following examples, where no specific conditions are mentioned, are carried out according to standard methods and conditions, or according to the instructions of the commercial suppliers.

[0103] SFC chiral separation method in the following examples:

[0104] Enantiomeric compound I was obtained using the following chiral separation conditions:

[0105] Chromatographic column: Chiralpak AD-3.50 A, 4.6 mm, inner diameter 3 um;

[0106] Mobile phase: A: CO2, B: EtOH (0.05% diethanolamine);

[0107] Gradient: MPB from 5% to 50% in 1.2 min, hold for 1 min, then 0.8 min from 50% to 5%;

[0108] Flow rate: 3.4 mL / min;

[0109] Column temperature: 35 °C;

[0110] ABPR: 1800 psi.

[0111] RT of enantiomeric compound I: 1.445 min.

[0112] Example 1: Preparation of compound I

[0113]

[0114]

[0115] To a 500 mL reaction flask, IPA (100 mL), concentrated HCl (100 mL), a- acetonaphthone (55.0 g), benzylmethylamine (40 g), and paraformaldehyde (17.2 g) were added sequentially with stirring. The temperature was raised to reflux, approximately 95 °C, with stirring. After 7.5 h at reflux, the reaction was monitored by HPLC and was complete. The reaction was concentrated to dryness, and ethyl acetate was added. The mixture was stirred overnight. The mixture was filtered, and the filter cake was washed with ethyl acetate until the filtrate was colorless. The solid was dissolved in DCM, and the mixture was stirred until a suspension formed. The pH was adjusted to ~9 with aqueous NaHC03, and the mixture was partitioned. The organic phase was dried and concentrated to give compound 3 as a light yellow oil, 75.0 g, 76% yield. 1H NMR (400 MHz, CDCl3) δ 8.58(d, J = 8.5 Hz, 1H), 7.94 (d, J = 8.2 Hz, 1H), 7.85 (d, J = 7.9 Hz, 1H), 7.80(d, J = 7.1 Hz, 1H), 7.54 (ddd, J = 14.9, 14.5, 7.3 Hz, 2H), 7.47 – 7.41 (m,1H), 7.25 – 7.17 (m, 5H), 3.50 (s, 2H), 3.24 (t, J = 7.1 Hz, 2H), 2.91 (t, J= 7.1 Hz, 2H), 2.22 (s, 3H)。

[0116] Into a 500 mL reaction flask, add THF (180 mL), TMP (24.90 g), and stir to cool to -72 °C. Start temperature-controlled dropwise addition of n-BuLi (12.46 g), and the system becomes a milky white suspension. After about 2 h of temperature-controlled reaction, start temperature-controlled dropwise addition of compound 4 (36.4 g), and after the dropwise addition is complete, temperature-controlled reaction for 2 h, and monitor the reaction, with no obvious starting material. Start temperature-controlled dropwise addition of 3 (42.78 g), and after 3 h of temperature-controlled reaction, send a sample for monitoring, and the reaction is complete. Add the reaction solution to 200 mL of saturated NH4Cl solution, and wash twice. Wash the organic phase with brine once, and separate the phases. Dry and concentrate the organic phase, and then stir the sample to pass through a column to obtain compound 5 as a colorless oil.

[0117] Into a 500 mL reaction flask, add 5 (50.0 g) and DCM (250 mL), stir to dissolve, and then add chloroformic acid-1-chloroethyl ester (11.6 g), and stir at room temperature for 3 h. Concentrate the reaction solution to dryness, then add methanol, and continue stirring at room temperature for 2 h. After the reaction is complete as monitored by HPLC, directly add an aqueous NaHCO3 solution to quench. After quenching to pH ~ 9, add DCM, separate the phases, and dry and concentrate the organic phase. Stir the solid sample to pass through a column to purify, and obtain a light yellow solid, which is subjected to SFC chiral resolution to obtain compound I. RT: 1.445, ee value: 100%, purity: 98.93%, yield: 18%. 1H NMR (400 MHz, DMSO) δ 8.61 (d, J = 7.8 Hz, 1H), 8.49 (s, 1H), 8.41 (s, 1H), 8.24 (s, 1H), 7.92 (d, J = 6.6 Hz, 2H), 7.71 (d, J = 8.1 Hz, 5H), 7.62 - 7.50 (m, 4H), 7.34 (t, J = 7.5 Hz, 1H), 7.17 (d, J = 5.7 Hz, 2H), 5.72 (s, 1H), 4.14 (s, 4H), 2.81 (s, 1H), 2.32 (s, 1H), 2.08 (s, 4H), 1.99 (s, 1H).

[0118] Example 2

[0119]

[0120]

[0121] Into a 500 mL reaction flask, add IPA (100 mL), concentrated HCl (100 mL), a-acetylnaphthalene (55.0 g), compound 6 (90.1 g), and paraformaldehyde (17.2 g) sequentially with stirring. Stir to warm to reflux, which is about 95 °C. After refluxing for 7.5 h, monitor the reaction by HPLC. Concentrate to dryness, then add ethyl acetate and stir overnight. Filter, and wash the filter cake with ethyl acetate until the filtrate is colorless. Add DCM to the solid. After stirring to form a suspension, adjust the pH to ~9 with aqueous NaHC03, and separate the layers. Dry and concentrate the organic phase to give oily liquid compound 3-1, which totals 115.6 g with a yield of 77%.

[0122] Into a 500 mL reaction flask, add IPA (100 mL), concentrated HCl (100 mL), a-acetylnaphthalene (55.0 g), compound 6 (90.1 g), and paraformaldehyde (17.2 g) sequentially with stirring. Stir to warm to reflux, which is about 95 °C. After refluxing for 7.5 h, monitor the reaction by HPLC. Concentrate to dryness, then add ethyl acetate and stir overnight. Filter, and wash the filter cake with ethyl acetate until the filtrate is colorless. Add DCM to the solid. After stirring to form a suspension, adjust the pH to ~9 with aqueous NaHC03, and separate the layers. Dry and concentrate the organic phase to give oily liquid compound 3-1, which totals 115.6 g with a yield of 77%. 1H NMR (400 MHz, CDCl3) δ 8.53 (d,J = 8.5 Hz, 1H), 7.93 (d, J = 8.2 Hz, 1H), 7.80 (d, J = 7.9 Hz, 1H), 7.72(d,J = 7.1 Hz, 1H), 7.51 (ddd, J = 14.9, 14.5, 7.3 Hz, 2H), 7.46-7.48 (d, J=7.5 Hz, 6H ), 7.45 – 7.41 (m, 1H), 7.25 – 7.21 (m, 6H), 7.18 – 7.14 (t, J =7.2 Hz, 14.4 Hz, 3H), 3.27 (t, J = 7.1 Hz, 2H), 2.84 (t, J = 7.1 Hz, 2H),2.24 (s, 3H)。

[0123] Into a 500 mL reaction flask, THF (180 mL), TMP (24.90 g), stirring and cooling to -72 °C. Start temperature control dropwise n-BuLi (12.46 g), the system into a white suspension. Temperature control reaction for about 2 h, temperature control dropwise compound 4 (36.41 g), after the end of the dropwise, temperature control reaction 2 h, monitoring the reaction, no obvious raw material. Start temperature control dropwise 3-1 (64.24 g), temperature control reaction for three hours, monitoring, reaction is completed. The reaction solution is added to 200 mL of saturated NH4Cl solution, washed twice. Organic phase is washed with brine once, and separated. The organic phase is dried and concentrated, and then stirred to pass through a column to obtain colorless oily compound 5-1.

[0124] Into a 500 mL reaction flask, 5-1 (62.4 g), DCM (250 mL), stirring and dissolving, then adding chloroformic acid-1-chloroethyl ester (11.7 g), stirring at room temperature for 3 h, the reaction solution is concentrated to dryness, then adding methanol, continue to stir at room temperature for 2 h. HPLC monitoring reaction is completed, directly adding NaHCO3 aqueous solution to quench. To pH ~ 9 quenching, adding DCM, washing and separating. The organic phase is dried and concentrated, and then stirred to pass through a column to obtain yellow solid, SFC chiral resolution to obtain compound I. RT: 1.445, ee value: 100%, purity: 98.94%, yield 19%. 1H NMR (400 MHz, DMSO) δ 8.61 (d, J = 7.8 Hz, 1H), 8.49(s, 1H), 8.41 (s, 1H), 8.24 (s, 1H), 7.92 (d, J = 6.6 Hz, 2H), 7.71 (d, J =8.1 Hz, 5H), 7.62 - 7.50 (m, 4H), 7.34 (t, J = 7.5 Hz, 1H), 7.17 (d, J = 5.7Hz, 2H), 5.72 (s, 1H), 4.14 (s, 4H), 2.81 (s, 1H), 2.32 (s, 1H), 2.08 (s,4H), 1.99 (s, 1H).

[0125] Example 3

[0126]

[0127]

[0128] Into a 500 mL reaction flask, add methanol (100 mL) and methylamine (17.94 g) under stirring, cool to 20 °C, add 2,4-dimethoxybenzaldehyde (80 g) in batches. React at room temperature for 2 hours, add sodium borohydride (21.87 g) in batches, monitor the reaction by HPLC, adjust the pH to weak acidity with hydrochloric acid, concentrate, dissolve in ethyl acetate, wash with sodium chloride, separate, concentrate to obtain compound 7, total 61.1 g, yield 70%.

[0129] Into a 500 mL reaction flask, add IPA (100 mL) and concentrated hydrochloric acid (100 mL) under stirring, add α-acetylnaphthalene (55.0 g) and compound 7 (58.6 g), add polyformaldehyde (17.2 g). Stir and heat to reflux, about 95 °C. After refluxing for 7.5 h, monitor the reaction by HPLC. Concentrate to dryness, then add ethyl acetate, stir overnight. Filter, wash the filter cake with ethyl acetate until the filtrate is colorless. Add DCM to the solid. After stirring to form a suspension, adjust the pH to about 9 with sodium bicarbonate aqueous solution, separate. Dry and concentrate the organic phase to obtain compound 3-2 in the form of an oily liquid, total 89.2 g, yield 76%. 1H NMR (400 MHz, CDCl3) δ 8.52 (d,J = 8.5 Hz, 1H), 7.91 (d, J = 8.2 Hz, 1H), 7.80(d, J = 7.9 Hz, 1H), 7.72 (d,J = 7.1 Hz, 1H), 7.50 (dd, J = 14.9, 14.5, 7.3 Hz, 2H), 7.46 – 7.39 (m, 2H),6.43 (d, J = 4.4 Hz, 1H), 6.31 (s, 1H), 3.81 (s, 6H), 3.56 (s, 2H), 3.23 (t,J = 7.1 Hz, 2H), 2.91 (t, J = 7.1 Hz, 2H), 2.26 (s, 3H)。

[0130] Into a 500 mL reaction flask, THF (180 mL), TMP (24.90 g), stirred and cooled to -72 °C. Started to control temperature dropwise to add n-BuLi (12.46 g), the system became a white suspension. After about 2 h of temperature control reaction, control temperature dropwise to add compound 4 (36.4 g), after the end of dropwise, control temperature reaction 2 h, monitor the reaction, no obvious raw material. Started to control temperature dropwise to add compound 3-2 (51.3 g), after three hours of temperature control reaction, sent sample monitoring, reaction was completed. The reaction solution was added to 200 mL of saturated NH4Cl solution, washed twice. The organic phase was washed with brine once again, and separated. The organic phase was dried and concentrated, and then stirred to pass through a column to obtain colorless oily compound 5-1.

[0131] Into a 500 mL reaction flask, compound 5-2 (50.0 g), DCM (250 mL), stirred and dissolved, then added chloroformic acid-1-chloroethyl ester (11.6 g), stirred at room temperature for 3 h, the reaction solution was concentrated to dryness, then added methanol, continued to stir at room temperature for 2 h. After HPLC monitoring reaction was completed, directly added NaHCO3 aqueous solution to quench. After quenching to pH ~ 9, added DCM, washed and separated. The organic phase was dried and concentrated, and then stirred to pass through a column to obtain yellow solid, SFC chiral resolution to obtain compound I. RT: 1.445, ee value: 100%, purity: 98.95%, yield 17%. 1H NMR (400 MHz, DMSO) δ 8.61 (d, J = 7.8 Hz,1H), 8.49 (s, 1H), 8.41 (s, 1H), 8.24 (s, 1H), 7.92 (d, J = 6.6 Hz, 2H), 7.71(d, J = 8.1 Hz, 5H), 7.62 - 7.50 (m, 4H), 7.34 (t, J = 7.5 Hz, 1H), 7.17 (d,J = 5.7 Hz, 2H), 5.72 (s, 1H), 4.14 (s, 4H), 2.81 (s, 1H), 2.32 (s, 1H), 2.08(s, 4H), 1.99 (s, 1H).

[0132] Example 4

[0133]

[0134]

[0135] Into a 500 mL reaction flask, IPA (100 mL), concentrated HCl (100 mL), a- acetonaphthone (55.0 g), p-methoxybenzylmethylamine (46 g), and paraformaldehyde (17.2 g) were added sequentially with stirring. The temperature was raised to reflux, ca. 95 °C, with stirring. After 7.5 h at reflux, the reaction was monitored by HPLC and was complete. The reaction was concentrated to dryness, and ethyl acetate was added. The mixture was stirred overnight. The solid was filtered, and the filter cake was washed with ethyl acetate until the filtrate was colorless. The solid was taken up in DCM, and the suspension was stirred. The pH was adjusted to ~9 with aqueous NaHCO3, and the mixture was partitioned. The organic phase was dried and concentrated to give compound 3-3 as an oil, 83.4 g, 75% yield. 1 H NMR (400 MHz, CDCl3) δ8.53 (d, J = 8.5 Hz, 1H), 7.93 (d, J = 8.2 Hz, 1H), 7.80(d, J = 7.9 Hz, 1H),7.72 (d, J = 7.1 Hz, 1H), 7.51 (dd, J = 14.9, 14.5, 7.3 Hz, 2H), 7.48 – 7.21(m, 3H), 6.45 (d, J = 4.4 Hz, 2H), 3.83 (s, 3H), 3.56 (s, 2H), 3.22 (t, J =7.1 Hz, 2H), 2.94 (t, J = 7.1 Hz, 2H), 2.26 (s, 3H).

[0136] Add 180 mL of THF and 24.90 g of TMP to a 500 mL reaction flask, stir, and cool to -72 °C. Begin adding n-BuLi (12.46 g) dropwise under controlled temperature, resulting in a milky white suspension. After approximately 2 hours of controlled temperature reaction, add compound 4 (36.4 g) dropwise under controlled temperature. After the addition is complete, continue the reaction under controlled temperature for 2 hours, monitoring the reaction; no obvious starting material is observed. Begin adding 3-3 (47.1 g) dropwise under controlled temperature. After 3 hours of controlled temperature reaction, send a sample for monitoring; the reaction is complete. Add the reaction solution to 200 mL of saturated NH4Cl solution and wash twice. Wash the organic phase once more with brine and separate the layers. After drying and concentrating the organic phase, stir and pass through a column to obtain a colorless oily substance.

[0137] Add 53g of methyl chloroformate (MCC) and 250mL of dimethyl chloroformate (DMC) to a 500mL reaction flask. After stirring and dissolving, add 11.6g of 1-chloroethyl chloroformate. Stir at room temperature for 3 hours. Concentrate the reaction solution to dryness, then add methanol and continue stirring at room temperature for 2 hours. After the reaction is completed as monitored by HPLC, quench it directly with NaHCO3 aqueous solution. After quenching to pH ~9, add DCM, wash, and separate the solution. Dry and concentrate the organic phase, then purify the solid by column chromatography to obtain a pale yellow solid. Chiral resolution by SFC yields compound I. RT: 1.445, ee: 100%, purity: 98.94%, yield: 19%. 1 H NMR (400 MHz, DMSO) δ 8.61 (d, J = 7.8 Hz, 1H), 8.49 (s,1H), 8.41 (s, 1H), 8.24 (s, 1H), 7.92 (d, J = 6.6 Hz, 2H), 7.71 (d, J = 8.1Hz, 5H), 7.62 – 7.50 (m, 4H), 7.34 (t, J = 7.5 Hz, 1H), 7.17 (d, J = 5.7 Hz,2H), 5.72 (s, 1H), 4.14 (s, 4H), 2.81 (s, 1H), 2.32 (s, 1H), 2.08 (s, 4H), 1.99 (s, 1H).

[0138] Example 5

[0139] 1. Objective: To test the in vitro activity of compound I against clinical isolates of Mycobacterium tuberculosis (including drug-sensitive and drug-resistant strains).

[0140] 2. Experimental materials:

[0141] Controlled drugs: Compound II was provided by Shanghai Jiatan Pharmaceutical Technology Co., Ltd.; the compound II was compound I-1 in PCT / CN2017 / 070835 example 1; isoniazid (INH), rifampicin (RFP), moxifloxacin (MFX) and PA-824 were purchased from Sigma-Aidrich; bedaquiline (BDQ) was purchased from Shanghai Hanshang Biological Technology Co., Ltd.;

[0142] Strains: Strain / H37Rv (ATCC 27294), strain / ATCC 30248, strain / ATCC 30031, strain / ATCC 30129, strain / ATCC 30050, strain / ATCC 30091, strain / ATCC 30144, strain / ATCC 30180, strain / ATCC 30611, strain / ATCC 29065, strain / ATCC 30797, strain / ATCC 30390, strain / ATCC 29928, strain / ATCC 29925, strain / ATCC 30105, strain / ATCC 30586, strain / ATCC 28198, strain / ATCC 30367, strain / ATCC 30102, strain / ATCC 30149, strain / ATCC 30577, strain / ATCC 27830 were provided by the National Tuberculosis Clinical Laboratory of Beijing Chest Hospital (Beijing Chest Tuberculosis and Tumor Institute), Capital Medical University, and the strains were preserved in the Drug Research Laboratory.

[0143] Culture medium: 7H9 liquid medium (containing 10% OADC) was a product of BD company.

[0144] 3. Experimental method

[0145] The minimum inhibitory concentration (Minimum Inhibitory Concentration, MIC) of compound I, compound II, bedaquiline (BDQ), isoniazid (INH), rifampicin (RFP), moxifloxacin (MFX) and PA-824 on 5 strains of Mycobacterium tuberculosis clinical isolates sensitive strains and 15 strains of Mycobacterium tuberculosis clinical isolates drug-resistant strains was determined by using microplate Alamar Blue method (MABA).

[0146] The microplate Alamar Blue method (MABA) was determined according to Lu Yu, Wang Bin, Zheng Meiqin, et al. Application of Alamar Blue and MTT to determine the minimum inhibitory concentration of anti-tuberculosis drugs. Chinese Journal of Tuberculosis Prevention and Control, 2007, 29 (6): 499-501.

[0147] (1) Accurately weigh compound I and the control drug. Dissolve isoniazid (INH) in sterile distilled water. Dissolve compound I, compound II, rifampin (RFP), moxifloxacin (MFX), PA-824 and bedaquiline (BDQ) in dimethyl sulfoxide (DMSO) to prepare a high-concentration stock solution (see Table 1). Dilute with 7H9 liquid medium to the concentration of the application solution. The final concentration of compound I and the control drug in the medium is twice the concentration. The final concentrations for the strains are as follows: compound I: 32~0.0156μg / mL; compound II: 32~0.0156μg / mL; INH: 40~0.0195μg / mL; RFP: 40~0.0195μg / mL; MFX: 20~0.0097μg / mL; PA-824: 40~0.0195μg / mL; BDQ: 10~0.005μg / mL.

[0148] Table 1. Preparation of Compound I and Control Drug

[0149]

[0150] (2) Strains culture and inoculation

[0151] Each clinical isolate was treated with Difo containing 10% OADC. TM Incubate in Middlebrook 7H9 medium at 37°C with 5% CO2 for 2-3 weeks until the logarithmic growth phase, then freeze at -80°C. Remove the cryopreserved solutions of each clinical strain from the -80°C storage cabinet and dilute each strain to 1*10-1. 6 CFU / mL, pipette 100μL and add sequentially to the drug-containing wells and bacterial control wells of a 96-well plate, bringing the final volume of each well to 200μL. The final inoculum concentration is 5*10⁻⁶. 5 CFU / mL.

[0152] (3) Cultivation and Result Observation

[0153] The 96-well plates were incubated at 37°C with 5% CO2 for 7 days. After 7 days, Alamar Blue was added. The process was performed in a biosafety cabinet in the dark. Alamar Blue was mixed evenly at a ratio of 20 μL to 12.5 μL of 20% Tween 80 per well, and 32.5 μL was added to each well using an eight-channel pipette. At this point, all wells were blue. The plates were then incubated at 37°C with 5% CO2 for another 24 hours, and the color change was observed. The fluorescence value of Alamar Blue in the 96-well plates was measured using a multi-functional microplate reader (Tecan Infinite m200) at wavelengths (560 nm, 590 nm). The minimum inhibitory concentration (MIC) of each drug was calculated by observing the color change and fluorescence value.

[0154] 4. Experimental results and conclusions

[0155] The MIC data of compound I and control drugs against 5 clinical isolates of sensitive strains and 15 clinical isolates of drug-resistant strains in this experiment are shown in Tables 2 and 3.

[0156] Table 2 MIC results (ug / ml) of 5 sensitive Mycobacterium tuberculosis clinical strains

[0157]

[0158] The MIC ranges of compound I and control drugs against 5 sensitive Mycobacterium tuberculosis clinical strains were 0.142-0.273 ug / ml; compound II: 0.073-0.107 ug / ml; BDQ: 0.018-0.019 ug / ml; INH: 0.029-0.038 ug / ml; RFP: 0.068-0.091 ug / ml; MFX: 0.035-0.056 ug / ml; PA-824: 0.077-0.288 ug / ml. The activity of compound I against 5 sensitive Mycobacterium tuberculosis clinical strains was similar to that of compound II, BDQ, INH, RFP, MFX, and PA-824.

[0159] Table 3 MIC results (ug / ml) of 15 drug-resistant Mycobacterium tuberculosis clinical strains

[0160]

[0161] The MIC results of compound I and control drugs against 15 drug-resistant Mycobacterium tuberculosis clinical strains showed that, except for inhibiting strains / ATCC 30144 and strains / ATCC 30586, the bacteriostatic activity of compound I against the other 13 drug-resistant Mycobacterium tuberculosis clinical strains was similar to that of compound II, BDQ, and PA-284. INH and RFP did not show activity against 15 drug-resistant Mycobacterium tuberculosis clinical strains, and MFX showed activity against some of the 15 drug-resistant Mycobacterium tuberculosis clinical strains.

[0162] Conclusion: Compound I has good in vitro activity against Mycobacterium tuberculosis clinical isolates, including sensitive strains and drug-resistant strains, and the activity is similar to that of compound II and BDQ, and there is no cross-resistance with other anti-tuberculosis drugs.

[0163] Example 6

[0164] 1. Drug

[0165] Compound I and compound II were provided by Shanghai Jiatan Pharmaceutical Technology Co., Ltd.;

[0166] 2. Administration method

[0167] Postprandial administration (MAD): The subjects were admitted to the Phase I clinical trial ward on the day before the corresponding test, and the unified life management was prohibited. Any food or beverage other than the unified diet was prohibited. The subjects ate dinner around 18:00, and then fasted without water. On the first day of single-dose administration, on the first day of multiple-dose administration, and on the 8th day, the subjects ate a regular meal around 8:00, which was completed within 30 minutes; 30 minutes after starting the meal, the subjects were given the test drug (active drug or placebo) by the researchers, and 240 ml of water was used to take the medicine. Water was prohibited for 1 hour after taking the medicine. Lunch and dinner were served at 4 hours and 10 hours after taking the medicine, respectively.

[0168] 3. Pharmacokinetic evaluation

[0169] Multiple dosing: blood samples were collected at 0 h (within 60 minutes before administration), 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 12 h, 24 h, 48 h, 72 h (D4 before administration), 96 h (D5 before administration), 120 h (D6 before administration), 144 h (D7 before administration), 264 h (D12 before administration) before the first administration, and 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 264 h after the last administration.

[0170] 4. Method validation of bioanalysis and sample testing

[0171] The test drug concentration detection was conducted by Shanghai Xihua Detection Technology Co., Ltd. The concentration of Compound II and Compound I in human plasma was determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS), and the internal standard method was used for quantification. The plasma sample used was 100 μL, the lowest limit of quantification (LLOQ) of Compound II in plasma was 10.0 ng / mL, and the detection range was 10.0-5000 ng / mL; the lowest limit of quantification (LLOQ) of Compound I was 0.100 ng / mL, and the detection range was 0.100-50.0 ng / mL.

[0172] In order to ensure the reliability of the established LC-MS / MS method for determining the concentration of Compound II and Compound I in plasma, the method was completely validated. The method validation results met the acceptance criteria of ICH M10 and other relevant guidelines for linearity, accuracy, precision, selectivity (interference), recovery, matrix effect, dilution reliability, residual during batch sample testing process, batch capacity, stability, etc.

[0173] After the method validation met the requirements, the test samples were determined. Quality control samples of different concentrations were prepared to evaluate the analysis method.

[0174] 5. Pharmacokinetic parameter calculation

[0175] According to the blood concentration of each subject, the pharmacokinetic parameters of the plasma parent compound II and compound I were calculated by Phoenix WinNonlin 8.1 software using a non-compartment model.

[0176] The specific calculation formula is as follows: t1 / 2z = ln2 / λz.

[0177] Wherein, t1 / 2z: terminal elimination half-life; λz: elimination rate constant. The absolute value of the slope of the terminal segment of the semi-logarithmic drug-time curve calculated by linear regression method.

[0178] 6. The experimental results are shown in Table 4

[0179] Table 4

[0180]

[0181] As can be seen from the above table data, the human half-life of compound I is much longer than that of compound II. After reaching steady state, it can be administered once a week, which is convenient for patients to take medicine and overcomes the heavy burden of finished medicine due to the multi-drug use of patients with pulmonary tuberculosis.

[0182] Example 7

[0183] 1. Purpose: Test the in vitro activity of compound I on laboratory-induced BDQ-resistant strains

[0184] 2. Experimental materials:

[0185] Control drug: Compound II is provided by Shanghai Jiatan Pharmaceutical Technology Co., Ltd.; the compound II is PCT / CN2017 / 070835 example 1 compound I-1; bedaquiline (BDQ) is purchased from Shanghai Hanshang Biological Technology Co., Ltd.;

[0186] Strains: Strain / H37RV, strain / BDQ-resistant strain 1, strain / BDQ-resistant strain 2, strain / BDQ-resistant strain 3 are provided by the National Tuberculosis Clinical Laboratory of Capital Medical University Affiliated Beijing Chest Hospital (Beijing Tuberculosis and Thoracic Tumor Institute), and the strains are preserved in the drug research room.

[0187] Culture medium: 7H9 liquid medium (containing 10% OADC), which is a product of BD company.

[0188] 3. Experimental method

[0189] The Alamar Blue (MABA) microplate assay was used to determine the minimum inhibitory concentration (MIC) of compound I, bedaquiline, isoniazid (INH), and rifampin (RFP) against laboratory-induced BDQ-resistant strains.

[0190] The determination of the minimum inhibitory concentration of anti-tuberculosis drugs by microplate Alamar Blue method (MABA) is based on the research of Lu Yu, Wang Bin, Zheng Meiqin et al., on the determination of the minimum inhibitory concentration of anti-tuberculosis drugs by Alamar Blue and MTT. Chinese Journal of Antituberculosis, 2007, 29(6): 499-501.

[0191] (1) Accurately weigh compound I and the control drug. Compound I, compound II and bedaquiline (BDQ) were dissolved in dimethyl sulfoxide (DMSO) to prepare a high-concentration stock solution (see Table 5). The solution was diluted with 7H9 liquid medium to the concentration of the application solution. The final concentration of compound I and the control drug in the medium was twice the concentration. The final concentrations for the strains were as follows: compound I: 32~0.0156 μg / mL; compound II: 32~0.0156 μg / mL; BDQ: 10~0.005 μg / mL.

[0192] Table 5. Preparation of Compound I and Control Drug

[0193]

[0194] (2) Strains culture and inoculation

[0195] Various laboratories induced BDQ-resistant strains in Difo containing 10% OADC. TM Incubate in Middlebrook 7H9 medium at 37°C with 5% CO2 for 2-3 weeks until the logarithmic growth phase, then freeze at -80°C. Remove the cryopreserved solutions of each clinical strain from the -80°C storage cabinet and dilute each strain to 1*10-1. 6 CFU / mL, pipette 100μL and add sequentially to the drug-containing wells and bacterial control wells of a 96-well plate, bringing the final volume of each well to 200μL. The final inoculum concentration is 5*10⁻⁶. 5 CFU / mL.

[0196] (3) Cultivation and Result Observation

[0197] The 96-well plate is placed in a 5% CO2 incubator at 37°C for 7 days. After 7 days, add Alamar Blue, operate in a biosafety cabinet in the dark, mix 20 μL of Alamar Blue and 12.5 μL of 20% Tween 80 in each well, then use an eight-channel pipette to add 32.5 μL to each well. At this time, the color of each well is blue, continue to place in a 5% CO2 incubator at 37°C for 24 hours, then observe the color change. Use a multifunctional enzyme label instrument (Tecan infinite m200) to measure the fluorescence value of Alamar Blue in the 96-well plate at wavelengths (560 nm, 590 nm), and calculate the minimum inhibitory concentration of each drug by observing the color change and fluorescence value.

[0198] 4. Experimental results and conclusions

[0199] In this experiment, the MIC data of compound I and the control drug on the laboratory-induced BDQ-resistant strain are shown in Table 6.

[0200] Table 6 MIC results of laboratory-induced BDQ-resistant strains (ug / ml)

[0201]

[0202] The MIC results of compound I and the control drug on the laboratory-induced BDQ-resistant strain show that compound I has good in vitro activity on BDQ-resistant strain 1 and BDQ-resistant strain 2, which is better than compound II and BDQ, and there is no cross-resistance with other anti-tuberculosis drugs.

[0203] Example 8

[0204] First, the effects of compound I and compound III on the action potential of Purkinje fibers in New Zealand white rabbits were studied.

[0205] 1. Positive control: haloperidol (purchased from SIGMA)

[0206] Test sample: compound I (Shanghai Jiatan Pharmaceutical Technology Co., Ltd.) and compound III (Wuhan Pharmaron New Drug Development Co., Ltd.);

[0207] Negative control group: 0.3% DMSO (purchased from China National Pharmaceutical Group);

[0208] 2. Preparation and analysis of drug preparation

[0209] (1) Preparation method of drug preparation stock solution

[0210] Negative control: measure the required volume of DMSO into the container as stock solution;

[0211] Test article: Under the condition of light protection, appropriate amount of compound I and compound III were weighed, and appropriate amount of DMSO was added to prepare 10 mmol / L stock solution. The prepared solution was placed in a container and stored at room temperature in the dark.

[0212] Positive control: Appropriate amount of haloperidol was weighed, dissolved in appropriate amount of DMSO to prepare 1 mmol / L stock solution, and the prepared solution was stored at -20°C for standby use.

[0213] (2) Preparation method of working solution of administration preparation

[0214] Before action potential determination, the DMSO and compound I group administration preparations were diluted with appropriate amount of Tyrode's solution to the required concentration of working solution, and the positive control group administration preparation was taken out from the -20°C refrigerator, melted and diluted with appropriate amount of Tyrode's solution to the required concentration of working solution. The specific method is as follows in Table 7:

[0215] Table 7

[0216]

[0217] 3. Experimental method

[0218] (1) Experimental animals

[0219] New Zealand white rabbits, SPF level; 40; 2.4-3.5 kg, 3-5 months old; purchased from China Food and Drug Inspection Research Institute.

[0220] (2) Preparation of isolated tissues

[0221] 1) The test animals were anesthetized (3% sodium pentobarbital, about 45-60 mg / kg, ear vein injection), the abdomen was cut open, the chest was opened, the heart was exposed, and the heart was removed.

[0222] 2) The heart was quickly placed in a about 37°C Tyrode's liquid container saturated with 95% O2+5% CO2 mixed gas, and the heart was gently squeezed to expel the remaining blood in the ventricle. The Tyrode's liquid (mmol): NaCl 137, KCl 4, MgSO4·7H2O 1, NaH2PO4·2H2O 0.4, NaHCO3 12, CaCl2 1.8, Glucose 5.6, PH 7.35-7.4, 95% O2+5% CO2 mixed gas saturated for more than 30 min;

[0223] 3) After the remaining blood in the ventricle is discharged, the heart is quickly placed in a 2-8 ℃ high potassium (27 mM) Tyrode's liquid vessel saturated with 95% O2+5% CO2 mixed gas to stop the heart, wherein the high potassium (27 mM) Tyrode's liquid (mmol): NaCl 137, KCl 27, MgSO4·7H2O 1, NaH2PO4·2H2O 0.4, NaHCO3 12, CaCl2 1.8, Glucose 5.6, PH 7.35-7.4, 95% O2+5% CO2 mixed gas saturated for more than 30 min.

[0224] 4) Under a microscope, the Purkinje fibers are carefully separated.

[0225] (3) Sample addition

[0226] 1) Sample addition approach: use WPI peristaltic pump to add working solution to the dish where the New Zealand white rabbit Purkinje fibers are located, and use Harvard micro-perfusion tank to ensure that the outlet temperature of the peristaltic pump perfusion needle is 36±2℃.

[0227] 2) Sample addition group sequence and sample addition time

[0228] Test sample working solution: from low concentration to high concentration (0.3, 1, 3 and 10 μmol / L (in Tyrode's solution)), after about 15 min of sample addition, the effect reaches steady state, and a higher concentration of test sample working solution is added.

[0229] DMSO: Add negative control working solution (0.3% DMSO (in Tyrode's solution)) before adding test sample working solution, and after about 5 min of sample addition, the effect reaches steady state, and a low concentration of test sample working solution can be added.

[0230] Positive control working solution: select different tissues to be tested to add positive control working solution (1 μmol / L haloperidol (in Tyrode's solution)).

[0231] 3) Parameter recording

[0232] Resting potential (RMP); action potential 0 phase rising speed (Vmax); action potential amplitude (APA); action potential duration (APD)

[0233] 4. Data processing and statistical analysis

[0234] (1) Data processing

[0235] The experimental data analysis is completed by Lab Chart 7.3.8 software. The last 5 action potentials recorded before and after sample addition of each tissue are selected for analysis.

[0236] RMP, APD, APA and Vmax were expressed as mean ± standard deviation, and the percentage of data change before and after sample addition was calculated. The average of RMP, APD, APA and Vmax of each concentration group of test sample / control sample was corrected by the average of RMP, APD, APA and Vmax before sample addition of each tissue, and the percentage change (△%) of RMP, APD, APA and Vmax of each concentration group was calculated, as follows:

[0237] The percentage change (△%) of RMP, APD, APA or Vmax = [(A-B) / B] x 100%

[0238] A: RMP, APD, APA or Vmax of each concentration group of test sample / control sample

[0239] B: RMP, APD, APA or Vmax before sample addition of the same tissue

[0240] (2) Statistical analysis

[0241] Software used: SPSS Statistics 21.

[0242] RMP, APD, APA, Vmax and the percentage change of the above parameters were statistically analyzed. When each concentration group of test sample was compared with the negative control group, the following methods were used for statistics: ① Levene's test was used to detect the homogeneity of variance of data. When the variance was homogeneous (P > 0.05), one-way analysis of variance (ANOVA) was performed. When the variance was not homogeneous (P≤0.05), Dunnett T3 test was used for inter-group comparison test (0.05 and 0.01 levels). ② When the results of variance analysis were statistically different (P≤0.05), Dunnett t test was used for inter-group comparison test (0.05 and 0.01 levels). When the results of variance analysis were not statistically different (P > 0.05), the statistics ended.

[0243] When the positive control group was compared with DMSO, independent sample t test was used.

[0244] 5. Experimental results

[0245] Under the experimental conditions, the mean values and the percentage of change of RMP, APA, Vmax, APD60 and APD90 of Purkinje fiber action potential of New Zealand white rabbits were not obviously changed after administration of 0.3, 1, 3 and 10 μmol / L of compound I; the percentage of change of APD30 decreased after administration of 10 μmol / L of compound I, and the specific results are shown in Table 8; under the experimental conditions, the mean values of RMP, APA, Vmax, APD30, APD60 and APD90 and the percentage of change of RMP, APA, APD60 and APD90 of Purkinje fiber action potential of New Zealand white rabbits were not obviously changed after administration of 0.3, 1, 3 and 10 μmol / L of compound III, and the percentage of change of Vmax slightly increased; the percentage of change of APD30 of Purkinje fiber action potential of New Zealand white rabbits decreased after administration of 10 μmol / L of compound III, and the specific results are shown in Table 9.

[0246] Table 8 Effect of compound I on Purkinje fiber action potential of New Zealand white rabbits

[0247]

[0248] Note: the parameters are represented by mean ± standard deviation; n: number of tissues; P>0.05 compared with the 0.3% DMSO group.

[0249] Table 9 Effect of compound III on Purkinje fiber action potential of New Zealand white rabbits

[0250]

[0251] Note: the parameters are represented by mean ± standard deviation; n: number of tissues; P>0.05 compared with the 0.3% DMSO group.

[0252] 6. Experimental conclusion: the concentration of compound I affecting the parameters of Purkinje fiber action potential of New Zealand white rabbits is 10 μmol / L. The concentration of compound III affecting the parameters of Purkinje fiber action potential of New Zealand white rabbits is 0.3 μmol / L.

[0253] II. Effects of compound I and compound III on hCav1.2 channel

[0254] 1. Positive control: Nifedipine (purchased from Sigma);

[0255] Test sample: compound I (provided by Shanghai Jiatan Pharmaceutical Technology Co., Ltd.) and compound III (provided by Wuhan Pharmaron New Drug Development Co., Ltd.);

[0256] The structural formula of the compound III is as follows: ;

[0257] Negative control: DMSO (purchased from Sigma);

[0258] 2. Preparation and analysis of administration preparation

[0259] (1) Preparation method of administration preparation stock solution

[0260] The weighed compound I (10.07 mg) and compound III (10.04 mg) were prepared into about 29.941 mM and 29.969 mM stock solutions with DMSO;

[0261] (2) Preparation method of administration preparation working solution

[0262] 1) The test sample stock solution was sequentially diluted with DMSO to 0.01 mM, 0.033 mM, 0.1 mM, 0.33 mM and 1 mM;

[0263] 2) 30 μL of each concentration of the above test solution was added to 10 mL of detection external solution for dilution to prepare 0.03 μM, 0.1 μM, 0.3 μM, 1 μM and 3 μM solutions.

[0264] 3) Nifedipine was dissolved in DMSO to prepare a 50 mM stock solution, and the Nifedipine stock solution was sequentially diluted with dimethyl sulfoxide (DMSO) to 0.8 μM, 4 μM, 20 μM, 100 μM and 500 μM solutions.

[0265] 3. Experimental method

[0266] (1) Cell line

[0267] 1) A CHO cell line stably expressing hCav1.2 channels was used, and the hCav1.2 cells were constructed by the laboratory of Beijing Aisipu Biotechnology Co., Ltd.

[0268] 2) Maintenance medium: HAM'S F12 and 20% FBS.

[0269] 3) Cell passage: remove the old culture medium and wash once with PBS, then add 1 mL of 0.25% trypsin-EDTA solution, incubate at 37 °C for 1.5 minutes. When the cells are detached from the dish bottom, add 5 mL of 37 °C preheated complete culture medium.

[0270] The cell suspension was gently blown with a pipette to separate the aggregated cells. The cell suspension was transferred to a sterile centrifuge tube and centrifuged at 1000 rpm / min for 5 minutes to collect the cells. The cells were expanded or maintained by inoculating 2.5*105 cells (final volume: 10 mL) per 6 cm cell culture dish.

[0271] (2) Patch clamp detection

[0272] The voltage stimulation protocol for recording hCav1.2 channel current by whole-cell patch clamp is as follows: when the whole-cell patch is formed, the cell membrane voltage is clamped at -60 mV. The clamping voltage is removed from -60 mV to 0 mV (the specific voltage is determined by the pilot IV) and maintained for 0.3 seconds. Data is collected every 20 seconds to observe the effect of drugs on the peak value of hCav1.2 channel current. Experimental data is collected by EPC-10 amplifier (HEKA) and stored in PatchMaster (HEKA) software (software version: v2x73.2).

[0273] A capillary glass tube (BF150-86-10, Sutter Instruments) is drawn into a recording electrode using a microelectrode puller (P97, Sutter Instruments). Under an inverted microscope (AE31E, Motic), the recording electrode is contacted to the cell using a microelectrode manipulator (86PW420600, MCI Instruments) and negative pressure is applied to form a GΩ seal. After forming a GΩ seal, rapid capacitance compensation is performed, and then negative pressure is continuously applied to break the cell membrane and form a whole-cell recording mode.

[0274] After the hCav1.2 channel current of the whole-cell recording is stable, drug administration begins. Each drug concentration is tested for 5 minutes (or the current is stable) before the next concentration is tested. The cover glass with cells is placed in the recording bath above the lens of the inverted microscope, and the test compound and the external solution without the compound are sequentially flowed through the recording chamber from low concentration to high concentration by gravity perfusion method to act on the cells, and the liquid exchange is performed by vacuum pump in the recording. The current detected in the external solution without the compound is used as its own control group.

[0275] Multiple cells are independently tested repeatedly. All electrophysiological experiments are performed at room temperature.

[0276] 4. Data analysis

[0277] (1) Standardize the current after each drug concentration and the blank control current: Peak Currentcompound / Peak Current vehicle

[0278] (2) Calculate the inhibition rate corresponding to each drug concentration: 1-( Peak Current compound / Peak Current vehicle)

[0279] (3) The mean and standard error were calculated for each concentration, and all the values were calculated by Microsoft Excel 2013. In addition, the half inhibitory concentration of each compound was calculated by the following equation using IGOR software:

[0280] Inhibition rate = 1 / [1 + ( IC 50 / c) h ];

[0281] The above equation was used to fit the dose-dependent effect non-linearly, where c represents the drug concentration, IC 50 is the half inhibitory concentration, and h represents the Hill coefficient. Curve fitting and calculation of IC 50 were completed using IGOR software (software version: 6.0.1.0).

[0282] 5. Results

[0283] The inhibitory effects of Compound I and Compound III on hCav1.2 calcium channels were calculated, and the half inhibitory concentration (IC 50 ) of the sample on hCav1.2 calcium channel current was calculated by fitting, and the experimental results are as follows:

[0284] Table 10

[0285]

[0286] The experimental results of the positive control are as follows:

[0287] Table 11

[0288]

[0289] Table 12 Inhibitory effects of Compound I and Compound III on different ion channels

[0290]

[0291] Conclusion: In clinical practice, generally, rabbit Purkinje fiber assay AP concentration greater than 10 μM indicates no cardiovascular risk, and the inhibitory effect of hCav1.2 calcium channel greater than 3 uM represents the highest concentration of the method, indicating no cardiovascular risk; while the inhibitory effect of Compound III on hCav1.2 calcium channel is 0.75 and the rabbit Purkinje fiber assay AP concentration is 0.3, indicating a high cardiovascular risk.

[0292] Although the specific embodiments of the present application are described above, those skilled in the art should understand that these are only illustrative, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present application. Therefore, the scope of protection of the present application is defined by the appended claims.

Claims

1. A compound as shown in Formula I or a pharmaceutically acceptable salt thereof; 。 2. A compound as shown in Formula I-X or a pharmaceutically acceptable salt thereof; 。 3. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein, The retention time of the compound as shown in Formula I-X under the following SFC conditions is 1.445 min; Chromatographic column: Chiralpak AD-3.50A, 4.6 mm, inner diameter 3 um; Mobile phase: A: CO2, B: EtOH (0.05% diethanolamine); Gradient: MPB from 5% to 50% in 1.2 min, hold for 1 min, then 0.8 min from 50% to 5%; Flow rate: 3.4 mL / min; Column temperature: 35℃; ABPR: 1800 psi.

4. A compound as shown in Formula II, ； wherein R1 is an amino protecting group.

5. The compound of claim 4, wherein, R1 is -Bn, -Trt, -Dmb or -PMB.

6. The compound of claim 5, wherein, R1 is -Bn.

7. A compound as shown in Formula II-X, ； wherein R1 is an amino protecting group.

8. A method for preparing a compound of formula I, characterized in that, It comprises the following steps: In a solvent, the compound as shown in Formula II-X is subjected to a deprotection reaction in the presence of a deprotection reagent, and then subjected to chiral resolution to obtain the compound as shown in Formula I; ； wherein R1 is an amino protecting group.

9. The production method according to claim 8, wherein It meets one or more of the following conditions: 1) the solvent is dichloromethane; 2) the volume-mass ratio of the solvent to the compound as shown in Formula II-X is 3 mL / g~6 mL / g; 3) the deprotection reagent is chloroformic acid-1-chloroethyl ester and methanol; 4) the molar ratio of the deprotection reagent to the compound as shown in Formula II-X is 1.1:1~1.8:1; 5) the temperature of the deprotection reaction is room temperature; 6) the progress of the deprotection reaction is monitored by TLC, HPLC, GC or NMR; 7) the deprotection reaction comprises the following post-treatment steps: after the end of the deprotection reaction is monitored, the reaction is quenched, the PH is adjusted, and then extracted and separated, the obtained organic phase is concentrated and dried, and then subjected to column chromatography to obtain the compound as shown in Formula I-X.

10. The production method according to claim 9, wherein It meets one or more of the following conditions: 1) the volume-mass ratio of the solvent to the compound as shown in Formula II-X is 5 mL / g, 4 mL / g or 4.7 mL / g; 2) the molar ratio of the deprotection reagent to the compound as shown in Formula II-X is 1.4:1; 3) the progress of the deprotection reaction is monitored by HPLC.

11. A method of preparing a compound of Formula I comprising the steps of: In a solvent, the compound as shown in Formula II is subjected to a deprotection reaction as shown below in the presence of a deprotection reagent to obtain the compound as shown in Formula I; ； wherein R1 is an amino protecting group.

12. The production method according to claim 11, wherein It meets one or more of the following conditions: 1) the solvent is dichloromethane; 2) the volume-mass ratio of the solvent to the compound as shown in Formula II is 3 mL / g~6 mL / g; 3) the deprotection reagent is chloroformic acid-1-chloroethyl ester and methanol; 4) the molar ratio of the deprotection reagent to the compound as shown in Formula II is 1.1:1~1.8:1; 5) the temperature of the deprotection reaction is room temperature; 6) the progress of the deprotection reaction is monitored by TLC, HPLC, GC or NMR; 7) the deprotection reaction comprises the following post-treatment steps: after the end of the deprotection reaction is monitored, the reaction is quenched, the pH is adjusted, and then extraction and liquid separation are performed, the obtained organic phase is concentrated and dried, and then column chromatography is used for separation to obtain the compound shown in formula I.

13. The production method according to claim 12, wherein It meets one or more of the following conditions: 1) the volume-mass ratio of the solvent to the compound shown in formula II is 5 mL / g, 4 mL / g, or 4.7 mL / g; 2) the molar ratio of the deprotection reagent to the compound shown in formula II is 1.4:1; 3) the progress of the deprotection reaction is monitored by HPLC.

14. A method of preparing a compound of Formula II-X comprising the steps of: In the solvent, the compound shown in formula III is subjected to the reaction shown below with the compound shown in formula IV to obtain the compound shown in formula II-X; ； wherein R1 is an amino protecting group.

15. The production method according to claim 14, wherein It meets one or more of the following conditions: 1) the solvent is tetrahydrofuran; 2) the volume-mass ratio of the solvent to the compound shown in formula III is 4 mL / g to 6 mL / g; 3) the molar ratio of the base to the compound shown in formula III is 1.2:1 to 2:1; 4) the base is n-butyllithium; 5) the temperature of the reaction is -60°C to -80°C; 6) the preparation method comprises the following post-treatment steps: after the end of the reaction is monitored, the obtained reaction solution is added into a saturated NH4Cl solution, washed, extracted, and subjected to liquid separation, the obtained organic phase is washed with brine and then subjected to liquid separation, the obtained organic phase is concentrated and dried, and then column chromatography is used for separation to obtain the compound shown in formula II-X.

16. The production method according to claim 15, wherein It meets one or more of the following conditions: 1) the volume-mass ratio of the solvent to the compound shown in formula III is 5 mL / g; 2) the molar ratio of the base to the compound shown in formula III is 1.65:1; 3) the temperature of the reaction is -72°C.

17. Use of substance A or substance B in the preparation of a drug for inhibiting Mycobacterium tuberculosis; the substance A is the compound shown in formula I or a pharmaceutically acceptable salt thereof as claimed in claim 1; the substance B is the compound shown in formula I-X or a pharmaceutically acceptable salt thereof as claimed in claim 2 or 3.

18. The use of claim 17, wherein, It meets one or more of the following conditions: 1) the Mycobacterium tuberculosis is sensitive Mycobacterium tuberculosis or drug-resistant Mycobacterium tuberculosis; 2) the drug comprises a pharmaceutical excipient; 3) the substance A or the substance B is one or the only effective component of the drug; 4) the substance A is the compound shown in formula I; 5) the substance B is the compound shown in formula I-X; 6) the administration mode of the drug is oral; 7) the administration frequency of the drug is once a week.

19. The application as described in claim 18, characterized in that, It meets one or more of the following conditions: 1) the sensitive Mycobacterium tuberculosis is strain / ATCC 27830; 2) the drug-resistant Mycobacterium tuberculosis is bedaquiline-resistant Mycobacterium tuberculosis.

20. Use of a substance A or a substance B in the manufacture of a medicament for treating Mycobacterium tuberculosis infection; the substance A is a compound of Formula I or a pharmaceutically acceptable salt thereof as described in claim 1; the substance B is a compound of Formula I-X or a pharmaceutically acceptable salt thereof as described in claim 2 or 3.

21. The use of claim 20, wherein, which satisfies one or more of the following conditions: 1) the Mycobacterium tuberculosis is susceptible Mycobacterium tuberculosis or drug-resistant Mycobacterium tuberculosis; 2) the medicament comprises a pharmaceutical excipient; 3) the Mycobacterium tuberculosis infection is pulmonary tuberculosis; 4) the substance A or the substance B is one or the only effective component of the medicament; 5) the substance A is a compound of Formula I; 6) the substance B is a compound of Formula I-X; 7) the medicament is administered orally; 8) the medicament is administered once a week.

22. The use of claim 21, wherein, which satisfies one or more of the following conditions: 1) the susceptible Mycobacterium tuberculosis is strain / ATCC 27830; 2) the drug-resistant Mycobacterium tuberculosis is bedaquiline-resistant Mycobacterium tuberculosis.

23. A pharmaceutical composition comprising a substance A or a substance B, and a pharmaceutical excipient; the substance A is a compound of Formula I or a pharmaceutically acceptable salt thereof as described in claim 1; the substance B is a compound of Formula I-X or a pharmaceutically acceptable salt thereof as described in claim 2 or 3.

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